Polyphenols from green tea inhibit the growth of melanoma cells through inhibition of class I histone deacetylases and induction of DNA damage.

Prasad R, Katiyar SK - Genes Cancer (2015)

Bottom Line:
These effects of GTPs were associated with a significant inhibition of histone deacetylase (HDAC) activity, reduction in the levels of class I HDAC proteins, enhancement of histone acetyltransferase (HAT) activity and induction of DNA damage, as detected by Comet assay, in melanoma cells.GTPs-induced decrease in the levels of class I HDAC proteins is mediated through proteasomal degradation.Treatment of A375 and Hs294t cells with GTPs resulted in a decrease in the levels of cyclins and cyclin dependent kinases of G1 phase of cell cycle whereas upregulated the levels of tumor suppressor proteins (Cip1/WAF1/p21, p16 and p53).

Affiliation: Department of Dermatology, University of Alabama at Birmingham, Birmingham, AL, USA.

ABSTRACTMelanoma is the leading cause of skin cancer-related deaths. We have examined the effect of green tea polyphenols (GTPs), a natural mixture of epicatechin monomers, on melanoma cancer cell growth and the molecular mechanism underlying these effects using different human melanoma cell lines as an in vitro model. Treatment of melanoma cell lines (A375, Hs294t, SK-Mel28 and SK-Mel119) with GTPs significantly inhibited the cell viability as well as colony formation ability of melanoma cells in a dose-dependent manner. These effects of GTPs were associated with a significant inhibition of histone deacetylase (HDAC) activity, reduction in the levels of class I HDAC proteins, enhancement of histone acetyltransferase (HAT) activity and induction of DNA damage, as detected by Comet assay, in melanoma cells. GTPs-induced decrease in the levels of class I HDAC proteins is mediated through proteasomal degradation. Valproic acid, an inhibitor of HDACs, exhibited a similar pattern of reduced viability and induction of death of melanoma cells. Treatment of A375 and Hs294t cells with GTPs resulted in a decrease in the levels of cyclins and cyclin dependent kinases of G1 phase of cell cycle whereas upregulated the levels of tumor suppressor proteins (Cip1/WAF1/p21, p16 and p53).

Figure 3: Effect of GTPs on HDAC and HAT activity and the expression levels of class I HDACs proteins in melanoma cell linesA375 and Hs294t melanoma cell lines were treated with various concentrations of GTPs (0, 20, 40, and 60 μg/ml) for 24 or 48 h, and nuclear cell lysates were subjected to activity assays and western blot analysis. (A) The effect of GTPs on HDAC activity was measured using HDAC Activity Assay Kit, following manufacturer's instructions. Significant difference versus non-GTPs-treated control, *P<0.05, ¶P<0.01, †P<0.001. (B) Treatment of melanoma cell lines with GTPs for 48 h enhances HAT activity in cells. Significant difference versus non-GTPs-treated controls, ¶P<0.01, †P<0.001. Data are presented in terms of percent of control as mean ±SD, n=3. (C) Cells were treated with various concentrations of GTPs for 48 h, and cell lysates were subjected to western blot analysis for class I HDAC proteins. GTPs decrease the expression levels of HDAC proteins in melanoma cells. (D) GTPs enhance histone acetylation in melanoma cells when cells were treated for 48 h. Equal loading of protein samples was verified using anti-histone H3 antibody.

Mentions:
To determine the effect of GTPs on HDAC and HAT activities, two representative melanoma cell lines (A375 and Hs294t) were treated with various concentrations of GTPs (0, 20, 40, and 60 μg/ml) for 24 and 48 h. The nuclear fractions were subjected to the analysis of HDAC and HAT activities using their respective analytical kits. As shown in Figure 3A (left panel), GTPs treatment of A375 melanoma cells resulted in significant inhibition of HDAC activity (6-25% at 24 h, and 13-47% at 48 h; P<0.05 to P<0.001) as compared with vehicle-treated control cells and that this inhibitory effect of GTPs was dose- and time-dependent. Similar effects were also obtained when Hs294t cells were treated with GTPs (Fig. 3A, right panel). The effects of GTPs on HAT activity in A375 and Hs294t cells were determined using the HAT Activity Assay Kit. Treatment of cells with GTPs for 48 h resulted in significantly (P<0.01, P<0.001) higher levels of HAT activity in both A375 and Hs294t cells as compared to the control cells, which were not treated with GTPs, in a concentration-dependent manner (Figure 3B).

Figure 3: Effect of GTPs on HDAC and HAT activity and the expression levels of class I HDACs proteins in melanoma cell linesA375 and Hs294t melanoma cell lines were treated with various concentrations of GTPs (0, 20, 40, and 60 μg/ml) for 24 or 48 h, and nuclear cell lysates were subjected to activity assays and western blot analysis. (A) The effect of GTPs on HDAC activity was measured using HDAC Activity Assay Kit, following manufacturer's instructions. Significant difference versus non-GTPs-treated control, *P<0.05, ¶P<0.01, †P<0.001. (B) Treatment of melanoma cell lines with GTPs for 48 h enhances HAT activity in cells. Significant difference versus non-GTPs-treated controls, ¶P<0.01, †P<0.001. Data are presented in terms of percent of control as mean ±SD, n=3. (C) Cells were treated with various concentrations of GTPs for 48 h, and cell lysates were subjected to western blot analysis for class I HDAC proteins. GTPs decrease the expression levels of HDAC proteins in melanoma cells. (D) GTPs enhance histone acetylation in melanoma cells when cells were treated for 48 h. Equal loading of protein samples was verified using anti-histone H3 antibody.

Mentions:
To determine the effect of GTPs on HDAC and HAT activities, two representative melanoma cell lines (A375 and Hs294t) were treated with various concentrations of GTPs (0, 20, 40, and 60 μg/ml) for 24 and 48 h. The nuclear fractions were subjected to the analysis of HDAC and HAT activities using their respective analytical kits. As shown in Figure 3A (left panel), GTPs treatment of A375 melanoma cells resulted in significant inhibition of HDAC activity (6-25% at 24 h, and 13-47% at 48 h; P<0.05 to P<0.001) as compared with vehicle-treated control cells and that this inhibitory effect of GTPs was dose- and time-dependent. Similar effects were also obtained when Hs294t cells were treated with GTPs (Fig. 3A, right panel). The effects of GTPs on HAT activity in A375 and Hs294t cells were determined using the HAT Activity Assay Kit. Treatment of cells with GTPs for 48 h resulted in significantly (P<0.01, P<0.001) higher levels of HAT activity in both A375 and Hs294t cells as compared to the control cells, which were not treated with GTPs, in a concentration-dependent manner (Figure 3B).

Bottom Line:
These effects of GTPs were associated with a significant inhibition of histone deacetylase (HDAC) activity, reduction in the levels of class I HDAC proteins, enhancement of histone acetyltransferase (HAT) activity and induction of DNA damage, as detected by Comet assay, in melanoma cells.GTPs-induced decrease in the levels of class I HDAC proteins is mediated through proteasomal degradation.Treatment of A375 and Hs294t cells with GTPs resulted in a decrease in the levels of cyclins and cyclin dependent kinases of G1 phase of cell cycle whereas upregulated the levels of tumor suppressor proteins (Cip1/WAF1/p21, p16 and p53).

Affiliation:
Department of Dermatology, University of Alabama at Birmingham, Birmingham, AL, USA.

ABSTRACTMelanoma is the leading cause of skin cancer-related deaths. We have examined the effect of green tea polyphenols (GTPs), a natural mixture of epicatechin monomers, on melanoma cancer cell growth and the molecular mechanism underlying these effects using different human melanoma cell lines as an in vitro model. Treatment of melanoma cell lines (A375, Hs294t, SK-Mel28 and SK-Mel119) with GTPs significantly inhibited the cell viability as well as colony formation ability of melanoma cells in a dose-dependent manner. These effects of GTPs were associated with a significant inhibition of histone deacetylase (HDAC) activity, reduction in the levels of class I HDAC proteins, enhancement of histone acetyltransferase (HAT) activity and induction of DNA damage, as detected by Comet assay, in melanoma cells. GTPs-induced decrease in the levels of class I HDAC proteins is mediated through proteasomal degradation. Valproic acid, an inhibitor of HDACs, exhibited a similar pattern of reduced viability and induction of death of melanoma cells. Treatment of A375 and Hs294t cells with GTPs resulted in a decrease in the levels of cyclins and cyclin dependent kinases of G1 phase of cell cycle whereas upregulated the levels of tumor suppressor proteins (Cip1/WAF1/p21, p16 and p53).